One-Step Cloning

Cloning Made Easy Using Type IIS Restriction Endonucleases

Modular cloning and expression of complex genetic assemblies are becoming widely popular. Recent applications based on site-specific and homologous recombination have substantially improved the efficiency and reduced the time needed to obtain the final genetic construct. However, due to their intrinsic recombination mechanism, these strategies fail to operate with repetitive sequences or they leave unwanted sequences behind. Traditional approaches, such as standard restriction cloning, also result in assemblies with scars flanking the cloned fragments.

Method’s Principle

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Figure 1. Overview of the cloning strategy: (A) Diagram of the seamless assembly of two fragments using a combination of type IIS restriction enzymes and ligase. The fragments and their corresponding sequences at their ends are shown in blue and green. Adaptors added to the fragments are shown in red. Underlined characters represent the recognition site for the type IIS restriction endonuclease BsaI. (B) Diagram showing the cloning of two PCR fragments into a vector (left) and the transfer and assembly of fragments from two donor plasmids into a vector (right). The black arrows indicate the orientation of the restriction sites, starting at the restriction site and pointing toward the cleavage site. (C) Diagram showing the transfer of a single fragment from a donor plasmid into multiple vectors with varying features geared toward different downstream applications. Arrows and abbreviations are as in (B). (D) Diagram of the recipient vector pType-IIS. CamR, chloramphenicol resistance gene; Amp, ampicillin resistance gene; Kan, kanamycin resistance gene; ccdB, ccdB counterselectable marker.

During the past few years a novel approach based on the use of type IIS restriction enzymes in combination with T4 DNA ligase has become popular (known as Golden Gate cloning, PLoS ONE 3, e3647, 2008). Briefly, the type IIS restriction enzymes, a subset of type II restriction endonucleases, have the particularity of cleaving the DNA a few base pairs away from the recognition site. Recognition sites are strategically placed at the ends of the fragments that one intends to clone in such a way that upon DNA cleavage: i) exogenous sequences including the restriction site are removed, and ii) compatible overhangs are generated (Figure 1A).

Once ligated, the fragments become immune to further digestion by the restriction enzyme as the recognition sites have been eliminated. Thus, the restriction and ligation reactions can be consolidated into a single reaction. In the final construct the junctions between any pair of adjacent fragments carry no added or deleted sequences, thereby representing a true scarless assembly.

The system can be utilized i) for the seamless assembly of multiple fragments into a vector in a predetermined order, using PCR fragments or plasmids as donors (Figure 1B), or ii) for transferring fragments from a central repository donor clone into different customized vectors geared toward different applications (Figure 1C).

Three Blends

The sequence-dependent nature of the approach requires certain restriction sites to be absent from the fragments to assemble. To maximize the applicability of the system we optimized three different enzyme blends based on nonpalindromic recognition sites of varying lengths and GC-content: BbsI (6 bp, 50% GC), BsaI (6 bp, 66% GC), and AarI (7 bp, 71% GC). Each of the three blends contains all required enzymatic and nonenzymatic components and comes as a single 2X concentrated mix, thereby simplifying experimental design and minimizing pipetting. The mixes are provided with a recipient vector, pType-IIS, which serves as the seamless assembly backbone (Figure 1D).

Cloning Design Made Simple

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Figure 2. Assembly of different numbers and types of DNA fragments. (A) PCR Fragments were either i) precloned into pCR®-Blunt II- TOPO® and then assembled into 20 to 75 ng of pType-IIS or similar, or ii) assembled directly as PCR fragments into the same vectors as indicated. Twenty to 150 ng of each of the DNA fragments were employed. Reactions were either incubated for 1 h at 37°C or subjected to 30 cycles of [37°C for 1 min followed by 16°C for 1 min]. Aliquots were chemically transformed into DH10B™ or TOP10 One Shot® chemically competent cells. Experiments were performed, at least, in duplicate. (B) Cloning of repetitive or very small sequences. The indicated DNA fragments were assembled as in (A). The asterisk indicates that PCR fragments were directly used in the assembly reaction. Otherwise the DNA fragments were precloned into pCR®-Blunt II- TOPO®. TAL effector genes were assembled as proposed by Weber et al. (PLoS ONE, 6, e19722, 2011). (C) Short reaction times. A single 2 kb PCR fragment was cloned into pType-IIS using the indicated amount of DNA. Reactions were performed as in (A) for the indicated time and temperature.

Uncovering a viable solution for the assembly of one or multiple fragments into a vector using the seamless type IIS restriction endonuclease approach is not trivial as it requires, among other steps, positioning the recognition sites at appropriate places and in the right orientation so that the base complementarity of the overhangs at the end of each fragment triggers the self assembly of the expected construct. To facilitate the design, Life Technologies has made available a free webtool that takes the DNA sequence of up to 8 DNA fragments plus a recipient vector and returns the GenBank formatted sequence of the final assembled constructs, as well as the required oligonucleotides, and the individually modified DNA fragments needed for the assembly.

Cloning of Up to Eight Fragments

To test the efficiency of the three blends, multiple experimental designs were tried, employing different numbers and types of donor molecules of varying sizes. PCR-amplified fragments were either precloned into a TOPO vector or used as linear DNA fragments in the context of multi-component assemblies. The results showed that for 5 or more fragments, cloning efficiencies of 50% or higher could be obtained (Figure 2A).

Cloning of Repetitive and Very Small Sequences

One of the limitations of a few novel cloning approaches, such as those based on homologous recombination, is that they typically underperform or even fail to clone multiple repetitive sequences or DNA fragments with intrinsic particular properties. While in some cases the difficulty can be anticipated, such as with the assemblies of transcription activator-like (TAL) effector genes or alu elements, in other circumstances the cloning obstacles cannot be anticipated. The type IIS restriction approach overcomes some of these hindrances, as shown in Figure 2B. Repetitive sequences as those present in TAL effector genes or very short sequences can be readily assembled with fairly high cloning efficiencies.

Five-Minute Cloning

Simple assembly reactions—as, for example, those used to clone a single PCR fragment into a recipient vector—can be performed in 5 minutes with cloning efficiencies higher than 97% (Figure 2C). Longer incubation times result in an increase in the colony output.

Conclusions

The commercial tools presented here allow the researcher to seamlessly assemble multiple fragments into a vector in a predetermined way, circumventing the obstacles imposed by certain difficult-to-clone sequences. In addition, the intrinsic nature of the system permits the transfer of genes among different platforms. Therefore, the standardized unwanted sequences employed by other cloning approaches, which can interfere with downstream applications, are avoided with this method.

Lansha Peng, Xiquan Liang, Ph.D., and Chang-Ho Baek, Ph.D., are staff scientists; Jarrod Clark is a scientist III; and Federico Katzen, Ph.D. (federico.katzen@lifetech.com), is a senior manager, all at Life Technologies. Reagents referenced in this article (GeneArt® Type IIS Assembly Kits) are for Research Use Only. Not for human or animal therapeutic or diagnostic use. A free on-line tool that facilitates the cloning strategy and generates the required oligonucleotides can be found at bioinfo.invitrogen.com/oligoDesigner.

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